The picornavirus family, which includes encephalomyocarditis viruses (EMCV) have a genome which is composed of a single-stranded, positive sense RNA of about 7,500 to 8,300 nucleotides (Belsham et al., 1996). Picornavirus RNA is functionally monocistronic and, upon infection, is translated into a single polyprotein that is processed to yield structural and nonstructural virus proteins (Rueckert, R. R., 1996).
Picornaviral proteins and their precursors take their names (L, P1, P2, P3) from their sequential locations within the polyprotein. The leader or “L” proteins are present only in cardio- and aphthoviruses. The EMCV and Mengovirus leaders are about 7 kD in molecular weight. The four P1 peptides are the capsid proteins, 1A, 1B, 1C and 1D (EMCV: 8, 28, 25, and 30 kD). Those fragments derived from a common precursor stay together as a protomer unit throughout particle morphogenesis (Arnold et al., 1987; Grigera et al., 1985; Palmenberg, 1982). The middle portion of the polyprotein contains peptides 2A, 2B and 2C (EMCV: 16, 17, and 36 kD). Protein 2C is an ATPase (Pfister et al., 2000). In the entero- and rhinoviruses, 2C is also the genetic locus of the guanidine resistance marker, a compound that affects the initiation of RNA synthesis (Anderson-Sillman et al., 1984; Pincus et al., 1986).
However, the 2C protein is not a polymerase, and its contribution to the replication cycle remains unclear. The P3 peptides, 3A, 3BVPg, 3Cpro, and 3Dpol (EMCV: 10, 2, 22, and 51 kD) are more closely associated with genome replication. Preparations of 3Dpol can catalyze the elongation of nascent RNA chains in primer-dependent reactions, an activity that identifies this enzyme as a central element of viral polymerase complexes (Flanegan and Baltimore, 1977). Protein 3B is VPg, the peptide covalently linked to the 5′ end of the genome (Pallansch et al., 1980). VPg sequences are rich in basic, hydrophilic amino acids and have only one tyrosine residue (the attachment site) at position 3 from the amino end of the peptide. Initiation of positive- and negative-strand RNA synthesis requires VPg, perhaps as free protein or as part of a larger donor peptide (Morrow et al., 1984). Protease 3Cpro is the central enzyme in the viral cleavage cascade. After a co-translational primary break, catalyzed by a peptide cassette near the COOH-end of 2A (Hahn and Palmenberg, 2001), nearly all subsequent, or secondary cleavages within cardiovirus polyproteins, are affected by 3Cpro (Palmenberg, 1989).
Furthermore, infection with most picornaviruses is characterized by a strong inhibition of host cell protein synthesis at a time when virus-species proteins are efficiently produced (Ehrenfeld, E., 1996). Enteroviruses and rhinoviruses inhibit host translation, at least partially, by inactivation of eukaryotic translation initiation factor 4F (eIF4F), which binds to the cap structure of cellular mRNAs. eIF4F is composed of three polypeptides: eIF4E, eIF4A, and eIF4G. eIF4E is the cap-binding subunit (Sonenberg, N., 1996). Picornavirus RNAs are naturally uncapped and translate by a cap- and eIF4E-independent mechanism, by which the ribosomes bind to an (IRES internal ribosome entry site) (Agol, V. I., 1991).
Enteroviruses and rhinoviruses disrupt eIF4F through cleavage of the eIF4G subunit by 2Apro. This cleavage has been reported to be direct (Haghighat, et al., 1996) or indirect (Wyckoff et al., 1992). eIF4G cleavage does not preclude but, rather, stimulates cap-independent initiation of viral protein synthesis, since the cap-binding subunit, eIF4E, remains associated with the N-terminal cleavage product (Borman et al., 1997). The C-terminal cleavage fragment of eIF4G interacts with eIF4A and eIF3 to support IRES-dependent, but not cap-dependent, translation initiation (Borman et al., 1997).
In strong contrast to enteroviruses and rhinoviruses, it is widely known that no cleavage of eIF4G occurs following infection of cells with cardioviruses. Encephalomyocarditis virus, “EMCV” (NBCI Accession No. M81861), Mengovirus (NBCI Accession No. L22089), or Theilovirus (NBCI Accession Nos. M16020, M20562, and M20301) are examples of cardioviruses. It has been learned that these viruses have relatively efficient translation of their coding RNA in cell free systems. It has also been learned that there are various techniques for culturing and producing the natural cardioviruses.
Also, it is widely known that the 2A protein of EMCV is not similar to the enterovirus and rhinovirus 2Apro and does not possess protease consensus motifs or detectable proteolytic activity (Lloyd et al., 1988). Therefore, it is believed that cardiovirus infection does not induce eIF4G cleavage, and the encoded L and 2A proteins are not proteases. Furthermore, the genome sequences from EMCV, Mengo and the Theiler's viruses, which are known to encode 2A proteins (about 150 amino acids) do not share recognizable sequence similarity with any other viral or host proteins, including the 2As from other picornavirus species.
It has long been assumed that the shutoff of host cell protein synthesis after EMCV infection results from the ability of viral RNA to efficiently compete with capped cellular mRNAs for some limiting component of the translational machinery (Lawrence et al., 1974). Recently, it was suggested that EMCV causes the shutoff of host translation by dephosphorylation and activation of a suppressor of cap-dependent translation, 4E-BP1 (eIF4E-binding protein 1) (Gingras et al., 1996). However, to date no definitive mechanism has been established with respect to how EMCV causes the shutoff of host translation. Accordingly, it would be desirable to identify viral proteins involved in mRNA transcription and translation so as to enable the design of nucleic acid constructs which can be used to inhibit virally infected cells.